We have developed an intracellular immunization strategy with a genetically engineered human antibody clone as a novel antiviral therapy against the hepatitis C virus. This antibody clone targets the HCV NS3 protein, which has multiple enzymatic activities (protease, helicase and NTPase) that are crucial for viral genome replication. We have shown that a recombinant human antibody clone reacts with the helicase domain of HCV NS3 and completely inhibits the helicase activity. Intracellular expression of this antibody in either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. During the last review cycle of this application we have developed a mouse xenograft tumor model for HCV replication. We have shown that HCV replication in the subcutaneous tumors in the SCID mice is inhibited by interferon alpha. This provides a reliable animal model for testing intracellular immunization for HCV using the antibody. We also developed methods of encapsulation for plasmid DNA or purified antibodies into nanoparticles by using a biodegradable and FDA approved polymer. In this proposal we will be experimenting with the practical aspects of the intracellular immunization strategy in vitro as well as in vivo using a small animal model for hepatitis C virus. Our focus will be to improve technology for the systemic delivery of nanoparticles carrying the recombinant antibody to liver cells to inhibit HCV replication in a mouse model. Our hypothesis is that intracellular immunization with recombinant antibodies that block the NS3 helicase is an effective strategy for inhibiting hepatitis C virus replication and expression. We propose that encapsulation of the antibody gene into biodegradable nanoparticles will efficiently deliver the antibody gene to hepatocytes and may provide a novel therapeutic strategy for chronic HCV patients who are non-responders to interferon. In Specific Aim 1, we will define the epitope(s) of a recombinant human antibody clone and investigate what proportion of clinical HCV strains are inhibited by the recombinant human antibody clone. In Specific Aim 2, we will investigate the emergence of resistant virus variants that escapes from antibody-mediated inhibition of virus replication in HCV cell culture models. In Specific Aim 3, we will determine whether intracellular expression of recombinant antibody effectively eliminates virus RNA replication using a non-infectious small animal model for hepatitis C. If the experiments proposed in this grant application are successful, then we may have the basis for an alternative antiviral strategy for people with chronic HCV infections who do not respond to current interferon therapy. Public Health Relevance: Chronic hepatitis C virus infection accounts for 27% of liver cancer in the United States. This research proposal intends to develop an intracellular immunization strategy for inhibiting hepatitis C virus. If this project becomes successful then it can lead to a potential therapy to treat chronic hepatitis C infection and prevent liver cancer.